Hepatitis E virus (HEV) is the aetiological agent of non-HAV enterically transmitted hepatitis. It is the major cause of sporadic as well as epidemic hepatitis, which is no longer confined to Asia and developing countries but has also become a concern of the developed nations. In the Indian subcontinent, it accounts for 30-60% of sporadic hepatitis. It is generally accepted that hepatitis E is mostly self-limited and never progresses to chronicity. It has a higher mortality in pregnant women where the disease condition is accentuated with the development of fulminant liver disease. Currently, no antiviral drug or vaccine is licensed for HEV, although a vaccine candidate is in clinical trials. HEV genome is 7.2kb in size with three open reading frames (ORFs) and 5' and 3' cis acting elements, which have important roles to play in HEV replication and transcription. ORF1 codes for methyl transferase, protease, helicase and replicase; ORF2 codes for the capsid protein and ORF3 for a protein of undefined function. HEV has recently been classified in the genus Hepevirus of the family Hepeviridae. There are four major recognised genotypes with a single known serotype. The absence of a reliable in vitro propagation system is an obstacle to deciphering HEV biology. The genome of HEV has been cloned, sequenced and the infectious nature of these replicons has been established. However, questions related to replication, transcription, virus-host interactions and pathogenesis remain to be answered. This comprehensive review summarises the progress made so far in HEV research, and addresses some of the unanswered questions.

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1) is caused by a (CTG)n repeat expansion in the 3′ UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1). In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET) between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection.